Engineering human cell-based, functionally integrated osteochondral grafts by biological bonding of engineered cartilage tissues to bony scaffolds
Introduction
Articular cartilage is a highly specialized tissue, characterized by unique biomechanical properties and, on the other hand, a poor regenerative potential [1], [2]. As a consequence, when a traumatic lesion or a non-traumatic disease such as osteochondritis dissecans occurs, the defect is filled with a fibrous tissue which is not capable to withstand the high compressive and shear forces acting in the joint, often leading to the development of early osteoarthritis [1]. An established surgical technique for the repair of osteochondral (OC) defects (i.e., mosaicplasty) involves transplantation of autologous OC plugs consisting of an articular cartilage layer and the underlying subchondral bone [3], [4]. Despite the promising results reported, the use of autologous OC grafts suffers from several limitations, namely (i) amount of material available, (ii) donor site morbidity, and (iii) difficult graft/injured site matching. In vitro fabrication of OC composites of predefined size and shape starting from appropriate scaffolds, possibly combined with autologous cells, has the potential to overcome these limits [5].
To date, many tissue engineering approaches have been reported in literature to engineer OC grafts, both scaffold-free [6], [7], [8] or scaffold-based [9], [10], [11], [12] for the chondral layer and cell-free [12], [13], [14] or cell-seeded [10], [11], [15] for the bony layer. Also biphasic OC scaffolds have been proposed [15], [16], [17].
The clinical translation of such strategies however is limited by the fact that (i) animal cells are often used instead of human cells [6], [9]; (ii) the size of the graft is too small to be clinically relevant [6]; and (iii) the biomaterials used are not currently implanted in human patients. Moreover, the functionality of an engineered OC graft should be supported by (i) an efficient integration between the cartilage and bony layers, in order to withstand the shear forces acting in the joint, and at the same time (ii) a satisfactory maturation of the chondral layer, in order to allow partial loading and thus reduce post-operative immobilization. A chemical or physical bonding of the biomaterials would not necessarily guarantee a long lasting integration of the chondral and bony layers, especially if the degradation of the materials is not accompanied by an efficient neo-formation of extracellular matrix at the interface. Instead, a “biological bonding” between the two layers, namely their integration through the extracellular matrix synthesized by cultured cells, would provide an actively and durably interconnected interface even following the resorption/degradation of the biomaterials.
In this study, we aimed at developing and validating a method to engineer OC constructs based on the principle of biological bonding, and using clinically relevant cells and biomaterials. In particular, our strategy consisted in coupling a human adult chondrocytes (HAC)-seeded collagen type I/III matrix (Chondro-Gide®, Geistlich Pharma AG, Switzerland) with a cell-free devitalized bovine trabecular bone cylinder (Tutobone®, Tutogen Medical GmbH, Neuenkirchen am Brand, Germany) using fibrin glue (Tisseel®, Baxter Healthcare, Newbury, UK). Chondro-Gide®, Tutobone® and Tisseel® are extensively used biomaterials in clinical practice for cartilage repair, bone reconstruction and wound healing, respectively.
We opted for a cell-free bony layer since osteoprogenitor cells, abundantly present in the bleeding subchondral bone during implantation, are expected to colonize the scaffold and efficiently promote new bone formation [12], [16], [18]. We then developed a mechanical test and used it to address whether pre-culture time of chondrocyte-seeded matrices before combination with bony layers modulates the extent of integration between the two layers of resulting OC constructs. Finally, we investigated the quality of the chondral layer and whether it is affected by the presence of devitalized bone in culture, as previously described [9].
Section snippets
Cartilage biopsies, articular chondrocytes isolation and expansion
Cartilage tissues without signs of osteoarthritis were collected from the femoral condyles of 5 cadavers (3 males and 2 females; mean age: 57 years; range 31–81 years), following informed consent by relatives and in accordance with the Local Ethical Committee. Cartilage tissues were weighed, minced into small pieces and digested with 0.15% type II collagenase (10 mL solution/g tissue) for 22 h. The isolated human articular chondrocytes (HAC) were expanded for two passages with Dulbecco's Eagle's
Validation of peel-off tests for mechanical integration
Using cell-free OC constructs, we first tested two geometrical configurations, which based on the angle between the OC surface and the applied force are referred to as 90° peel-off or 180° peel-off (Fig. 2A). Both 90° peel-off and 180° peel-off were successfully implemented and gave repeatable results. Values measured with 90° peel-off were significantly higher than those measured with 180° peel-off (0.085 N ± 0.01 N and 0.065 N ± 0.01 N respectively; P < 0.05) (Fig. 2B). The 90° peel-off configuration
Discussion
In this study we succeeded in engineering in vitro functional osteochondral (OC) composites, where the chondral layer was efficiently developed and biologically integrated with a bony substrate through the extracellular matrix produced by human articular chondrocytes (HAC). In particular, in our experimental setting, which was based on clinically compliant cells and biomaterials, we demonstrated that 3 days of separate pre-culture of the chondral layer prior to its fusion with the bony layer
Conclusions
The OC grafts engineered according to the described method display several features advocating a large potential for clinical implementation, including: (i) a biological bonding of the chondral layer with the bony scaffold through the extracellular matrix produced by human cells; (ii) a clinically relevant diameter; (iii) a suitable stability allowing easy surgical handling and the possibility of insertion into the joint through a recently developed technique [32]; and (iv) a versatility in
Acknowledgments
The study was partially financed by the Swiss National Science Foundation (Grant No. 3200B0-110054), by the “Deutsche Arthrose-Hilfe e.V.” and by the Hardy & Otto Frey-Zünd Foundation. We gratefully thank Baxter Healthcare for kindly providing Tisseel® fibrin glue; Geistlich Pharma AG for the generous supply of Chondro-Gide® matrices; Tutogen Medical GmbH and Novomedics GmbH for the generous supply of Tutobone® scaffolds. We greatly thank Dr. Giuseppe Peretti for his fundamental scientific
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